Toxic L-tryptophan: Shedding Light on a Mysterious Epidemic—Problems with Identifying and Testing for Trace Contaminants
by William E. Crist
"I must emphasize that the presence of the contaminants in the [Showa Denko] L-tryptophan is astonishingly small and so you require very sophisticated instrumentation and a lot of hard work to even come close to determining the structures."
—Stephen Naylor, Mayo Clinic
Researchers have had enormous difficulty identifying the specific micro contaminant(s) in Showa Denko L-tryptophan that caused EMS.
In June 1990, the Los Alamos National Laboratory hosted a research conference on EMS in cooperation with the CDC, FDA, NIH and the New Mexico Department of Health and Environment. Phillip Hertzman, one of three New Mexico physicians who discovered the link between EMS and L-tryptophan consumption, summarized the conference proceedings in a paper published in the Journal of Rheumatology. Regarding toxicity tests on Showa Denko L-tryptophan, he and his colleagues stated:
"Radio-chemical studies have found no contamination. Analyses for 37 inorganic elements and for gross chemical contamination have been negative. Microbiological analyses have detected no significant contamination. While endotoxin has been detected in some lots, this contamination has not been associated with the illness. Several toxicological studies have been unable to produce the syndrome [EMS] in an animal model. Although the L-tryptophan was generally greater than 98.5% pure, high performance liquid chromatography analysis of material from the implicated manufacturer indicates the presence of more than 30 trace contaminants.
Other analyses such as ion chromatography and gel-permeation chromatography identified no case associated contaminants in Showa Denko product from the comparison of batches of control and case-associated L-tryptophan.
Using high performance liquid chromatography (HPLC), researchers have identified six case-associated contaminants in Showa Denko L-tryptophan. Two of these were novel amino acids: EBT (also called Peak 97 and Peak E) and 3-PAA (also called Peak I and UV-5). EBT and 3-PAA were significantly correlated to each other and patients with EMS ingested significantly greater amounts of both (10 and 15 times respectively) than did control L-tryptophan users.6Others reported, "The quantities of the known EMS case-associated contaminants, EBT and 3-PAA, were remarkably small, of the order of .01%, and could easily escape detection.; EBT and 3-PAA levels were 105 and 220 parts per million (ppm) respectively in one Showa Denko (SDK) L-tryptophan lot. Others reported 3-PAA levels at 89 ppm and 100 ppm, while EBT levels varied widely from zero to nearly 300 ppm.
In one study, Center for Disease Control (CDC) researchers found contaminant EBT in case-associated L-tryptophan lots dating back to August 19, 1986, more than two years before the alleged date of contamination. High levels of EBT were positively associated with EMS, but the association lacked statistical significance. The CDC researchers wrote: "While these findings do not rule out the possibility that EBT is the etiologic agent in EMS, they raise the possibility that other chemical contaminants in manufactured tryptophan modify the effects of EBT or that the causal agent of EMS is an entirely distinct compound.
Still, epidemiological data showed that EBT was the impurity most strongly associated with EMS and the Wilcoxon rank-sum test showed Peak 97 (EBT) was the single most predictive peak of case associated L-tryptophan lots.
Researchers said, "It is also possible that the causative agent (1) may not absorb in the UV [ultraviolet] range [used in chromatography] or (2) may be present in a peak that is hidden beneath another peak (e.g., the large L-tryptophan peak) on HPLC.
Rossanne Philen and Robert Hill at CDC commented on the biological potency of Showa Denko L-tryptophan trace contaminants:
"The L-tryptophan associated with EMS met the standards for purity established by the United States Pharmacopoeia (USP). Nevertheless, more than 60 micro contaminants have been found in L-tryptophan in amounts of 10 or fewer parts per million. This finding implies that if one of these 60 or more contaminants is the etiologic agent for EMS, the contaminant must be a biologically potent compound to cause such a serious disease at such a low dosage (emphasis added).
Animal Studies Fail to Reproduce Full EMS Pathology
An early study of implicated L-tryptophan (L-TRP) in Lewis rats produced encouraging results, replicating many of the pathological features of human EMS.
In late 1990, FDA and NIH researchers reported that Lewis rats treated with implicated L-TRP, but not USP grade L-TRP or vehicle control, developed many of the specific features of L-TRP EMS, including perimyositis, fasciitis, and perivascular inflammation. However, peripheral blood eosinophilia, a common feature of human EMS, was not observed. The large dosage of L-TRP given to the animals was equivalent to human consumption of about 5 grams/day of L-TRP (a recommended human dosage was 1-2 grams/day) and treatment was for 38 days.
"Our study provides evidence that the female Lewis rat is a suitable animal in which to study the effects of implicated L-tryptophan as well as the complex factors involved in the pathogenesis of inflammatory, fibrosing syndromes of muscle and fascia," the researchers concluded.
Douglas L. Archer, Ph.D., the deputy director of FDA's Center for Food Safety and Applied Nutrition, testified before a subcommittee hearing of the House of Representatives on July 18, 1991: "One of the most significant scientific breakthroughs was the development of an animal model for EMS…. After suspect LT [L-tryptophan] was fed to a special stain of rats, several pathological changes that were comparable to those in the EMS patients were observed."
But FDA's initial enthusiasm for an animal model of EMS was short lived.
In March 1993, government researchers led by Laurie Love, M.D., Ph.D., reported that while all animals (Lewis rats) treated with case-associated L-TRP or EBT developed significant myofascial thickening, compared with two control groups, even those animals receiving the control L-TRP showed a mild but significant increase in the thickness of the myofascia, compared with vehicle-treated control animals. The basic difference between this study and the previous one was that it was with a higher dosage (approx. 5-6 grams/day) of implicated L-TRP for a slightly longer period (42 days).
The study demonstrated for the first time the pathological effects of the EBT contaminant, but the results did not rule out the possibility that other impurities in the EMS-case-associated L-TRP might also contribute to some of the features of EMS. "Our study implicates EBT as one compound found in case-associated L-TRP that can cause some pathological changes in Lewis rats that are similar to some pathological features of L-TRP-associated EMS," according to the researchers.
Myofascial thickening and immune cell changes were most prominent in test animals after administration of case-associated L-TRP, but EBT when combined with control L-TRP was also found to cause immune cell activation in the peripheral blood.
The researchers stated, "This study also strongly suggests that control L-TRP alone [in high doses] plays an important role in this [EMS] and possibly other fibrosing illnesses, because it is associated with mild but significant myofascial thickening and alterations in peripheral mononuclear cell phenotypes, as well as with significant pancreatic pathology."
Edwin Kilbourne, M.D., a CDC epidemiologist, commented, "The meaning of the experimental findings in Lewis rats is not yet clear. The histopathologic changes produced by implicated tryptophan and by EBT mimic a part of the pathology of human EMS but fall short of reproducing the illness."
In 1998, a leading EMS researcher at the Mayo Clinic, Gerald Gleich, MD, updated the status of EMS research after scores of studies:
"The failure of both the bioassay and the animal feeding experiments to yield robust and reproducible results has been a major disappointment…. As a consequence of the investigations by public health authorities, L-tryptophan produced by Showa Denko KK has been implicated as a cause of EMS. Certain contaminants present in the L-tryptophan have been implicated as candidates for causation. However, we do not know the exact structures and without this knowledge one fears that another epidemic will occur at some time."
For more information on this subject, see also:
- National EMS Network Newsletter, winter 2000, interview with Stephen Naylor, researcher, Mayo Clinic.
- Herzman PA, et al., "The Eosinophilia-Myalgia Syndrome: The Los Alamos Conference," Journal of Rheumatology (1991), Vol. 18, No. 6, pp. 867-873.
- Toyo'oka T, et al., "Characterization of Contaminants in EMS-Associated L-tryptophan Samples by High-Performance Liquid Chromatography," Chem. Pharm. Bull. (1991), Vol. 39(3): pp. 820-822.
- Hill R, Caudill S, el al, "Contaminants in L-Tryptophan Assoicated with Eosinophilia Myalgia Syndrome,"Archives of Environmental Contamination and Toxicology (1993), Vol. 25, pp. 134-142.
- Arthur N. Mayeno, et al., "Characterization of 'Peak E," a Novel Amino Acid Associated with Eosinophilia-Myalgia Syndrome," Science (December 21, 1990), Vol. 25: pp. 1707-1708.
- Mayeno AN, et al., "3-(Phenylamino)alanine, a novel aniline-derived amino acid associated with the eosinophilia myalgia syndrome: a link to the toxic oil syndrome?" Mayo Clinic Proceedings (1992), Vol. 67, pp. 1134-1139.
- Mayeno AN, Gleich, JG, "Eosinophilia-Myalgia Syndrome and tryptophan production: a cautionary tale,"TIBTECH (Trends of Biotechnology) September 1994, Vol. 12, pp. 346-352.
- Simat T, et al., "Contaminants in biotechnologically manufactured L-tryptophan," Journal of Chromatography B (1996), Vol. 685, pp 41-51.
- Goda Y, et al., "3-anilino-L-alanine, structural determination of UV-5, a contaminant in EMS-associated L-tryptophan samples," Chem. Pharm. Bulletin (1992), Vol. 40(8), pp. 2236-2238.
- Philen, RM, et al., "Tryptophan Contaminants Associated with Eosinophilia-Myalgia Syndrome," American Journal of Epidemiology (1993), Vol. 138, No. 3, pp 154-159.
- Blauvelt A and Falanga V, "Idiopathic and L-Tryptophan-Associated Eosinophilic Fasciitis Before and After L-Tryptophan Contamination," Archives of Dermatology (August 1991), Vol. 127, pp. 1159-1166.
- Love LA, et al., "Pathological and Immunological Effects of Ingesting L-Tryptophan and 1,1'-Ethylidenebis (L-Tryptophan) in Lewis Rats," Journal of Clinical Investigation (March 1993), Vol. 91, pp. 804-811.
- MMWR, "Analysis of L-tryptophan for the Etiology of Eosinophilia-Myalgia Syndrome," (August 24, 1990), Vol. 39(34): pp. 589.
- Philen RM, et al., "3-(Phenylamino)alanine—a Link Between Eosinophilia-Myalgia Syndrome and Toxic Oil Syndrome?" Mayo Clinic Proceedings (1993), Vol. 68, pp. 197-200.
- Crofford L, et al., "L-Tryptophan Implicated in Human Eosinophilia-Myalgia Syndrome Causes Fasciitis and Perimyositis in the Lewis Rat," Journal of Clinical Investigation (November 1990), Vol. 86: pp. 1757-1763.
- Author's personal communications with EMS patients.
- Douglas L. Archer, Ph.D., Deputy Director, Center for Food Safety and Applied Nutrition, FDA, Hearing before the Subcommittee on Human Resources and Intergovernmental Relations Subcommittee of the Committee on Government Operations House of Representatives, First Session, July 18, 1991.
- Edwin M. Kilbourne, "Eosinophilia-myalgia syndrome: Coming to Grips with a New Illness," Epidemilogical Reviews, (1992), Vol. 4, pp. 16-36.
- Gerald J. Gleich, MD, "Current Status of Research on EMS," National EMS Network Newsletter, February (winter 1998).
© Copyright 2005 William E. Crist. All Rights Reserved